Nitrogenase and photosynthetic reaction centers catalyze electron transfer reactions required for the fundamental metabolic processes of biological nitrogen fixation and solar energy capture. The protein- cofactor complexes catalyzing these conversions are paradigms for the transduction of ATP and light energy in biological systems, as well as for the organization of electron transfer pathways. In addition to the specific mechanisms of nitrogenase and reaction centers, general similarities in the structural organizations of these complex electron transfer systems will be explored. Nitrogenase: The nitrogenase enzyme system catalyzes the ATP-dependent reduction of dinitrogen to the metabolically usable form of ammonia during biological nitrogen fixation. Nitrogenase is a prototypic example of an enzyme with multiple and varied iron-sulfur clusters that participate in electron transfer and substrate reduction, as well as providing an excellent model for energy transduction of ATP hydrolysis. The Fe-protein of nitrogenase is the obligate ATPase-dependent electron transfer agent for substrate reduction. To study how Fe-protein functions in this role, the following objectives will be investigated in the next grant period using a combination of crystallographic, mutagenesis, protein modification and nucleotide analogue approaches: l. Identification of the nucleotide binding sites in the Fe-protein. 2. Determination of the mechanism of ATP hydrolysis and the effects of ATP hydrolysis on the Fe-protein structure. Related time-resolved crystallographic studies on the reaction of phosphoglycerate kinase with ATP will also be performed. 3. Crystallographic determination of the structures of Fe-protein - MoFe- protein complexes, with emphasis on establishing how ATP hydrolysis and electron transfer are coupled. Photosynthetic reaction center: The bacterial reaction center (RC) is an integral membrane protein-cofactor complex that mediates charge separation across the membrane during the initial steps of photosynthesis. Mechanistic features to be addressed crystallographically include: 1. Analysis of site directed mutants altering the proton and electron transfer pathways. 2. Structure determination of the physiologically significant cytochrome c2 - RC complex. 3. Characterization of different RC redox states, including Laue studies after crystal illumination.